LABORATORIO DE URGENCIAS

 

 

 

Beta-Amyloid Production in Neurons and Astrocyte-Derived Cholesterol

 

                                                                                                Hao Wang, Joshua Kulas, Scott B. Hansen et al
                                                                                                                                   Sanford Consortium for Regenerative Medicine, La Jolla, CA

                                                                                                                      PNAS (August 2021)118 (33) e2102191118
                                                                                                                       https://doi.org/10.1073/pnas.2102191118

 

 

Alzheimer’s disease (AD), the most prevalent neurodegenerative disorder, is characterized by the progressive loss of cognitive function and the accumulation of amyloid β (Aβ) peptide and phosphorylated tau. Amyloid plaques are composed of aggregates of Aβ peptide, a small hydrophobic protein excised from the transmembrane domain of amyloid precursor protein (APP) by proteases known as beta- (β-) and gamma- (γ-) secretases
The nonamyloidogenic pathway involves a third enzyme, alpha- (α-) secretase, which generates a soluble APP fragment (sAPP-α), helps set neuronal excitability in healthy individuals, and does not contribute to the generation of amyloid plaques. Therefore, by preventing Aβ production, α-secretase–mediated APP cleavage reduces plaque formation. Strikingly, both pathways are finely regulated by cholesterol.
In cellular membranes, cholesterol regulates the formation of lipid clusters (also known as lipid rafts) and the affinity of proteins to lipid clusters, including β-secretase and γ-secretase.
α-secretase does not reside in lipid clusters; rather, α-secretase is thought to reside in a region made up of disordered polyunsaturated lipids. The location of APP is less clear. In detergent-resistant membrane (DRM) studies, it primarily associates with lipid from the disordered region, although not exclusively. Endocytosis is thought to bring APP in proximity to β-secretase and γ-secretase, and this correlates with Aβ production. Cross-linking of APP with β-secretase on the plasma membrane also increases Aβ production, leading to a hypothesis that lipid clustering in the membrane contributes to APP processing.
Testing this hypothesis in vivo has been hampered by the small size and transient nature of lipid clusters (often <100 nm), which is below the resolution of light microscopy. Superresolution imaging has emerged as a complimentary technique to DRMs, with the potential to interrogate cluster affinity more directly in a native cellular environment.
We recently employed superresolution imaging to establish a membrane-mediated mechanism of general anesthesia. In that mechanism, cholesterol causes lipid clusters to sequester an enzyme away from its substrate. Removal of cholesterol then releases and activates the enzyme by giving it access to its substrate.
In the adult brain, the ability of neurons to produce cholesterol is impaired. Instead, astrocytes make cholesterol and transport it to neurons with apolipoprotein E (apoE). Interestingly, apoE, specifically the e4 subtype (apoE4), is the strongest genetic factor associated with sporadic AD.
This led to the theory that astrocytes may be controlling Aβ accumulation through regulation of the lipid cluster function, but this has not yet been shown in the brain of an anima. We show that astrocyte-derived cholesterol controls Aβ accumulation in vivo and links apoE, Aβ, and plaque formation to a single molecular pathway.

RESULTS.

To establish a role for Aβ regulation by astrocytes in vivo, we first labeled and imaged monosialotetrahexosylganglioside1 (GM1) lipids in wild-type (WT) mouse brain slices. GM1 lipids reside in cholesterol-dependent lipid clusters and bind cholera toxin B (CTxB) with high affinity. These GM1 domains are separate from phosphatidylinositol 4,5 bisphosphate domains, which are polyunsaturated and cholesterol independent. We labeled GM1 domains (i.e., lipid clusters) from cortical slices with Alexa Fluor 647–conjugated, fluorescent CTxB and imaged with confocal and superresolution direct stochastical optical reconstruction microscopy (dSTORM). dSTORM is capable of visualizing nanoscale arrangements [i.e., sub–100-nm diameter lipid domain structures in intact cellular membranes.
CTxB appeared to label most cell types in cortical brain slices, green shading). In neurons, labeled with a neuron-specific antibody against neurofilament medium (NFM) chain protein, CTxB can be seen outlining the plasma membrane (outside of the cell), as opposed to NFM which labels throughout the cells.
Next, we investigated the role of cholesterol on the relative size and number of neuronal GM1 domains in brain tissue  using dSTORM (a ∼10-fold increase in resolution compared to confocal). In WT mouse cortical tissue, GM1 domains averaged ∼141 nm in apparent diameter, slightly smaller than the apparent size in primary neurons (∼150-nm diameter)). Cultured neuroblastoma 2a (N2a) cells exhibited the smallest clusters by far, on average only 100 nm in apparent diameter. All of the mammalian cells had domains larger than an intact fly brain, which had an apparent diameter of ∼90 nm. CTxB is pentadentate and can affect the absolute size; here, we used the numbers merely as a relative comparison of size under identical conditions.
To determine if astrocytes were a key cholesterol source regulating GM1 lipid domains, we compared the size of GM1 domains in neurons cocultured with cholesterol-deficient astrocytes. We depleted astrocyte cholesterol by SREBP2 gene ablation (SREBP2^−/−). SREBP2 is an essential regulator of cholesterol synthesis enzymes and was specifically knocked out in astrocytes using an ALDH1L1 promoter-driven Cre recombinase. We found that when cholesterol was depleted from astrocytes, the cluster size of primary neurons was significantly reduced (130 nm). The observed effect of astrocyte SREBP2 ablation on neurons suggests the decreased cholesterol transport to neurons—presumably through apoE.
To confirm that astrocytes could regulate neuronal GM1 domain formation through apoE, we added purified apoE to cultured primary neurons and N2a cells. The apoE (human subtype 3) derived from Escherichia coli was devoid of cholesterol, as prokaryotes do not make cholesterol. To provide a source of cholesterol to the apoE, we added 10% fetal bovine serum (FBS), a common source of mammalian lipids, including ∼310 µg/mL cholesterol-containing lipoproteins. ApoE can both load and unload cholesterol from cells, including neurons.
To confirm apoE’s effect in transporting cholesterol, we incubated apoE with N2a cells and analyzed the extracted lipids with mass spectrometry. The main apoE component extracted from the cells is cholesterol, including free cholesterol and cholesterol ester. Importantly, apoE is not present in FBS. Cells were treated acutely (1 h) with 4 µg/mL apoE, a physiologically relevant concentration seen in cerebral spinal fluid.
Loading cells with cholesterol (apoE and +FBS) caused an increase in the apparent cluster diameter from 100 to 130 nm in N2a cells. When cholesterol was unloaded, (ApoE absent a cholesterol source), the apparent size and number of GM1 domains decreased. Binning clusters by large >500 nm and small <150 nm showed a clear shift toward smaller domains, with apoE in low cholesterol, and a clear shift to large domains, with apoE in high cholesterol. We also measured cholesterol extraction by apoE in N2a cells using a quantitative absorbance assay. ApoE decreased the total cholesterol by ∼5%. To confirm this result, we compared apoE treatment to treatment with methyl β-cyclodextrin (MβCD), a nonnative chemical binder that extracts cholesterol from the plasma membrane and disrupts cluster function. MβCD caused a similar decrease, as seen for cells with cholesterol effluxed 

Superresolution Imaging of Amyloid-Processing Proteins in Lipid Clusters.

Next, we sought to characterize a cellular function of cholesterol loading To establish the movement of APP to each of its processing enzymes, α-, β-, and γ-secretases, directly in cellular membranes, we imaged N2a cells with dSTORM. APP was previously found in both cluster- and noncluster-like fractions of DRMs. DRMs are similar to GM1 lipid clusters. Imaging APP is important, since the amount of cluster-like association in DRMs could easily be affected by the detergent concentration used for preparation. We labeled GM1 domains with fluorescent CTxB and the amyloid proteins (APP, α-, β-, and γ -secretases) with Cy3b-labeled, fluorescent antibodies and determined cluster localization by pair correlation using density-based spatial clustering of applications with noise of two-color dSTORM images.

The Role of Astrocyte Cholesterol in Regulating APP Processing in Cultured Primary Neurons.

Since astrocytes regulate cholesterol levels and cluster formation in neurons, we hypothesized that astrocytes could directly control Aβ production in neuronal membranes. To test this hypothesis, we cultured primary cortical cells from mouse embryos (E17). In a first culture, we isolated just the neurons by sorting the cells with live cell fluorescence-activated cell sorting (FACS) with Thy1.2, a cell surface marker of mature neurons. In a second culture, we did not remove contaminating astrocytes from neurons, resulting in a mixed culture. All cultures were treated with or without apoE, fixed, and labeled with appropriate fluorophores; the neurons were then imaged with dSTORM. We confirmed the presence of astrocytes in our mixed culture by staining for glial fibrillary acidic protein (GFAP). About 1% of the mixed cell population was labeled by the GFAP antibody.
We found that the incubation of purified cortical neurons (Thy1.2⁺ with no astrocytes) with cholesterol-free human apoE dramatically decreased APP association with GM1 domains (>twofold) in primary neurons. However, when we mixed astrocytes with neurons, the same apoE had the exact opposite effect. GM1 domains sequestered APP, evident by a 2.5-fold increase in APP association with GM1 lipids. This suggests that apoE delivers cholesterol from the astrocytes to the neurons and increases GM1 domain affinity for APP. Similar imaging of the secretases showed that they remained immobile with α-secretase in the disordered region and β-secretase in GM1 domains. Thus, astrocyte-derived cholesterol is a potent signal that controls APP association with α- or β-secretase in the neuron, based on GM1 domain affinity.

The In Vivo Role of Astrocyte Cholesterol on Aβ and Plaque Formation in Mouse Brain.

Cholesterol is high in AD brains. Our model predicts that the attenuation of cholesterol in astrocytes should reduce the concentration of Aβ peptide formed in vivo. To test this hypothesis and to investigate the effect of astrocyte cholesterol on Aβ plaque formation in the intact brain, we crossed 3xTg-AD mice, a well-established mouse model with AD pathology, with our SREBP2^flox/flox GFAP–Cre mice to generate an AD mouse model lacking astrocyte cholesterol. In vivo Aβ exists in several forms, with Aβ1 to Aβ40 being the most abundant and Aβ1 to Aβ42 the most closely associated with AD pathology. The 3xTg-AD mouse expresses transgenes for mutant human APP and mutant presenilin 1 enzyme, resulting in a significant increase in human Aβ40 and Aβ42 production.
Using ELISAs, we measured human APP transgene-derived Aβ40 and Aβ42 in both the radioimmunoprecipitation assay buffer (RIPA) –soluble and –insoluble fractions in the hippocampus of aged control and transgenic AD mice. Hippocampus tissue from WT control mice was assessed to determine the nonspecific background of the Aβ signal in each ELISA. Knockout of SREBP2 in astrocytes of 3xTg-AD mice (AD × SB2^−/−) reduced soluble Aβ40 and Aβ42 levels in the hippocampus of 60-wk-old mice by twofold, to levels only slightly higher than WT controls, suggesting a near complete loss of amyloidogenic processing of APP. More impressively, insoluble Aβ40 and Aβ42 were almost entirely eliminated from the AD × SB2^−/− hippocampus. This was verified by immunofluorescence staining demonstrating an absence of amyloid plaques in AD × SB2^−/− mice. Of note, total APP transgene expression was not impacted by the loss of astrocyte SREBP2. Additionally, total GFAP and ApoE protein levels were not significantly changed by SREBP2 ablation. To further confirm that the reduced Aβ plaque burden in our model was due to reduced amyloidogenic processing, we performed mixed primary cultures of astrocytes and neurons from these 3xTg crosses. In agreement with our in vivo observation, we found that astrocyte SREBP2 deletion modestly increased sAPP-α production and robustly reduced sAPP-β generation without changing total APP abundance. The 3xTg-AD model also expresses a transgene for human tau protein, another key component of Alzheimer’s pathology. Tau is hyperphosphorylated in AD, and this is thought to be downstream of Aβ accumulation. It has also been proposed that cholesterol directly regulates tau phosphorylation. In agreement with both of these hypotheses, phosphorylation of tau at the key T181 residue, but not total tau, is eliminated in vivo in the AD × SB2^−/− hippocampus and reduced in mixed cultures in vitro. We also observed reduced brain tissue volume in AD × SB2^−/−animals, similar to that of astrocyte-specific SB2^−/− mouse brains characterized in our previous studies.

The Role of Astrocytic apoE in Regulating Membrane Cholesterol.

Although apoE is the major cholesterol transport protein in the brain, there are other proteins that may also contribute significantly to cholesterol transportation in the central nervous system. To test the effect of astrocytic apoE in transporting cholesterol, we treated neurons with astrocyte-conditioned media (ACM) from APOE knockout mice and compared the cholesterol loading effect with neurons treated with WT ACM. Specifically, we cultured neurons from SREBP2 GFAP–Cre mice to eliminate the transport of cholesterol from contaminating astrocytes in the culture. Neurons were treated with neurobasal media conditioned with astrocytes from WT or APOE^−/− animals. In Western blot, we verified that APOE^−/− ACM contains no apoE, while WT ACM does. Compared with control neurons without ACM treatment, WT ACM significantly increased APP’s association with GM1 clusters, while APOE^−/− ACM did not show any effect. This result again confirms that astrocytic apoE is required for cholesterol transportation from astrocytes to neurons. Other cholesterol transport proteins in the brain may allow some delivery of cholesterol, but apoE is the major protein for transporting cholesterol into neurons under these culture conditions.

DISCUSSION

Together, our data support astrocyte cholesterol as a key regulator of neuronal Aβ accumulation. The data from cultured neurons and astrocytes show that astrocyte-secreted apoE loads cholesterol into neurons. Increased cholesterol in neurons drives APP to associate with β- and γ-secretases in lipid clusters. The association of APP with lipid clusters appears to regulate the amount of Aβ accumulation, and Aβ levels dictate insoluble plaques.
While it has been known for some time that astrocytes play an important role in brain cholesterol production and express the AD-related protein apoE, the role of astrocytes in AD pathogenesis remains poorly understood. Astrocytes undergo robust morphological changes in neurodegenerative models, and recent research demonstrates that astrocytes undergo broad transcriptional changes early in the AD process. However, whether astrocytes are simply reacting to the AD neurodegenerative cascade or playing a role in promoting disease remains unclear. Here, we demonstrate a direct role for astrocytes in promoting the AD phenotype through the production and distribution of cholesterol to neurons. Combined, our data establish a molecular pathway that connects astrocyte cholesterol synthesis with apoE lipid trafficking and amyloidogenic processing of APP. The pathway establishes cholesterol as a critical lipid that controls the signaling state of a neuron. Cholesterol appears to be down-regulated in neurons as part of a mechanism to allow astrocytes to control APP presentation to proteolytic enzymes in the neuron. By keeping cholesterol low, the astrocyte can move the neuron through a concentration gradient that profoundly affects APP processing and eventual plaque formation. In essence, cholesterol is set up to be used as a signaling lipid. Rather than targeting a receptor, it targets GM1 domains and sets the threshold for APP processing by altering GM1 domain function. This concept is likely important to other biological systems, given the profound effect of cholesterol on human health.
The rise and fall of Aβ peptide with cholesterol is striking, and the data presented here support the proposed molecular mechanism in which amyloidogenic processing of APP is promoted by cholesterol increasing APP interactions with β- and γ-secretases through substrate presentation. This finding is in agreement with prior research, implicating increased brain cholesterol content as a factor which promotes AD-associated amyloid pathology. Administration of statins to guinea pigs significantly reduces Aβ levels in the cerebrospinal fluid, and statins reduced β secretase processing in induced pluripotent stem cell (iPSC) –derived neurons. APP/PS1 AD mice overexpressing a truncated active form of SREBP2 display an accelerated Aβ burden with aging. The conclusion is further supported by the existence of a cholesterol binding site in the APP protein. Our findings contribute to literature, demonstrating that amyloidogenic processing of APP in cultured cells occurs primarily in cholesterol-rich membrane domains and is consistent with cholesterol unloading decreasing Aβ formation in cell culture and on the plasma membrane.
Several lines of evidence suggest that cholesterol and its trafficking by lipoprotein particles also influences hyperphosphorylation of tau in neurons. van der Kant et al. found that, by disrupting cholesterol production using statins or enhancing cholesterol clearance, both pTau and total tau levels were reduced in cultured iPSC neurons. Interestingly, statins had no effect on neuronal pTau levels in the presence of proteasome inhibitors, suggesting that high levels of cholesterol in neurons may slow the intracellular clearance of tau protein. Moreover, we recently demonstrated that deletion of astrocyte apoE in a mouse model of tauopathy mitigates pTau pathology, neurodegeneration, and synapse loss, though total tau levels remained unchanged. This could indicate that distinct mechanisms exist for both exogenous and endogenous sources of cholesterol in promoting pTau accumulation in neurons. In a large study of Alzheimer’s pathology, APOE variants were shown to have strong effects on tau tangle burden, with APOE4/E4 carriers most adversely affected. Notably, a homozygous carrier of the APOE Christchurch mutation was reported to be resistant to cognitive decline and pTau accumulation, even in the presence of a presenilin 1 mutation and robust amyloid plaque deposition. Thus, the reduced levels of pTau we observe when astrocyte SREBP2 is ablated are likely driven by both reduced cholesterol levels as well as the alleviation of amyloid burden, though the exact mechanisms by which astrocyte cholesterol influences neuronal tau phosphorylation require future investigations.
The trend toward increased levels of cholesterol in apoE4 KI mice and the increased APP/GM1 clustering are consistent with the increased cholesterol in familial AD patients, which leads to the clustering of APP with lipid rafts and the increased Aβ production seen in neurons expressing apoE4 isoforms. We saw no difference on the ability of purified apoE3 and apoE4 to load or unload cholesterol into cultured cells; we conclude that cellular regulation of apoE, or protein abundance, is likely responsible apoE’s isoform-specific effects in vivo.
Cholesterol loading into glial cells is known to cluster inflammatory proteins and cause inflammation; hence, cholesterol in APP regulation suggests a correlative link between inflammation in AD and Aβ production. A previous study found gene expression–controlled Aβ production. We saw dramatic changes in Aβ production without any appreciable change to APP expression levels. Nonetheless, the two mechanisms are not mutually exclusive, and increased APP expression would be synergistic with increased cutting in response to high cholesterol.
At the molecular level, cholesterol loading regulates two functions: 1) the localization of APP to GM1 domains and 2) the total number of GM1 domains. Palmitoylation drives the cluster association of the majority of integral cluster proteins, but β- and γ-secretases are also palmitoylated and remain localized to GM1 lipids, suggesting a difference in relative affinity or additional factors that facilitate its release from GM1 domains [e.g., hydrophobic mismatch].
Many channels are palmitoylated and may respond to apoE and GM1 lipid domain function similar to APP. Investigating the effect of astrocyte cholesterol on channels will be important for studying AD, since many of the palmitoylated channels have profound effects on neuronal excitability and learning and memory. In separate studies, we saw that cholesterol loading with apoE caused the potassium channel TREK-1 to traffic to lipid clusters similar to APP—an effect that was reversed by mechanical force.
Our data suggest cultured cells lacking apoE supplementation likely underestimate physiological GM1 domain size, especially those derived from the brain where cholesterol is high. Not surprisingly, clusters appeared to be absent in cultured cells. Not until cholesterol, supplied by astrocytes or cholesterol-containing apoE, was added were nanoscale clusters present (∼100 nm). We conclude from our data that the conditions with cholesterol are the more physiologically relevant conditions for cultured cells.
All astrocyte-targeting Cre lines also delete to some degree in neural progenitor cells. While our model also suffers from this shortcoming, we have previously demonstrated that overall cholesterol synthesis by neurons increases to compensate for the loss of astrocyte cholesterol. Thus, the dramatic decrease in Aβ and pTau that we observe in our AD × SB2(^−/−) mice cannot readily be explained by the minority of knocked out neurons. In addition, microglia, which are also able to produce apoE and are not impacted by the Cre, are unable to compensate for a lack of astrocyte cholesterol synthesis. Our data emphasize that the small changes in Aβ production we see in our cell culture models may have a meaningful impact in vivo, such that the cumulative impact of altering cholesterol delivery to neurons can be observed.
We conclude that the availability of astrocyte cholesterol regulates Aβ production by substrate presentation. This contributes to the understanding of AD and provides a potential explanation for the role of cholesterol-associated genes as risk factors for AD.

Note: This presentation is part of the work. Tables, graphs, text, and complete references can be found in the publication mentioned above.

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